The present invention relates to an approach to cancer therapy which entails gene transfer in vivo.
Gene transfer has been recognized for some time as a promising avenue to therapies for cancers, among other diseases. The earliest applications of gene transfer for cancer treatment have been indirect approaches focusing on enhancing anti-tumor immune responses. Thus, for instance, attempts have been made to increase the cytotoxicity of immune cells, or to enhance their proliferation.
In one such approach, the gene for tumor necrosis factor (TNF) was inserted into tumor infiltrating lymphocytes (TIL) in an attempt to use the homing properties of TIL to deliver the toxic gene product preferentially to the tumor in situ. Initiation of this protocol has been difficult, however, because transduced T-cells shut down vector cytokine expression. Rosenberg et al., Human Gene Therapy 1: 443 (1990).
By another approach, tumor cells have been modified in vitro with cytokine genes and reintroduced into patients in an attempt to immunize the patient to their own cancer. In animal studies, the IL-4 gene was introduced to tumors by Tepper et al., Cell 57: 503 (1989); the IL-2 gene by Fearon et al., Cell 60: 397 (1990), and by Gansbacher et al., J. Exp. Med. 172: 1217 (1990); the interferon-gamma gene by Gansbacher et al., Cancer Res. 50: 7820 (1990); and TNF gene by Asher et al., J. Immunol. 146: 3227 (1991). Each of the animal studies demonstrated rejection of genetically altered tumors upon reimplantation, and the mice in these studies were immune to subsequent rechallenge with the same tumor.
Rosenberg and co-workers have carried out similar experiments in humans using the TNF gene and the IL-2 gene, respectively, with encouraging results. See Rosenberg et al., Human Gene Therapy 3: 57, 75 (1992).
These early investigations of the clinical use of gene transfer required that the tumor be excised and TIL or tumor cell lines established in culture which then could be gene-transduced in vitro and subsequently reimplanted into the patient. This approach is complicated, expensive, and limited by the fact that TIL and tumor lines cannot be regrown in vitro from the tumors of all patients and by the necessity to perform ex vivo transduction.
Retroviral vectors currently provide the most efficient means for ex vivo gene transfer in the clinical setting, but their usefulness has been seen as limited because retroviruses stably integrate only in target cells that are actively synthesizing DNA, and integration is a prerequisite to retroviral gene expression. Thus, attempts to use retroviruses to transfer genes into cell types that, as a population, are in G.sub.o, such as totipotent bone marrow stem cells, have had only limited success.
Cancers consist of actively replicating cells, however, and are often surrounded by quiescent normal cells. Thus, the above limitation may be exploited as an advantage in treating cancers, since a retroviral vector that carries a therapeutic agent would be integrated and expressed preferentially or exclusively in the cells of the cancerous mass.
In this regard Ezzeddine et al., New Biologist 3: 608-14 (1991), have reported on the use of retroviral vector-mediated gene transfer in vitro in an attempt to treat tumors. More specifically, a murine retroviral vector was employed to introduce a thymidine kinase gene from herpes simplex virus 1 ("HSV-1 tk gene") into C6 rat glioma-derived cell lines in vitro. Cells which had taken up the retroviral vector and expressed the tk gene were sensitized to the anti-viral agent ganciclovir, and were preferentially killed when exposed to ganciclovir in the medium.
Ezzeddine et al. were able to use the method to define conditions in vitro for killing essentially all infected cells but not uninfected cells. In addition, C6 cells were introduced subcutaneously into nude mice to form tumors and the tumor-bearing mice were treated with ganciclovir. Ganciclovir inhibited the growth of tumors formed by HSV-1 tk expressing C6 cells, but did not affect tumors formed by HSV-1 tk C6 cells.
Ezzeddine et al. thus showed that in vitro retroviral gene transfer can be used to sensitize cells to a cytotoxic agent, which can then be used to kill the cells when they are propagated as tumors in nude mice. The authors did not demonstrate any practical way to introduce an HSV-1 tk gene into tumor cells in situ, however. Ezzeddine et al. also did not show how to eradicate all neoplastic cells, a prerequisite for tumor remission, when less than all cells in the tumor would take up a tk gene, express the gene at a level sufficient to assure conversion of the drug to toxic and, as a consequence, be killed by exposure to ganciclovir.
Short et al., J. Neurosci. Res. 27: 427-33 (1990), have described the delivery of genes to tumor cells by means of grafting a retroviral vector-packaging cell line into a tumor. The packaging cell line produced a replication-defective retroviral vector in which the MoMLV LTR promoter-operator was used to drive expression of .beta.-galactosidase, which served as a marker of retroviral vector propagation. When the packaging cell line was grafted into a tumor, .beta.-galactosidase expression in situ was seen only in packaging cells and in proliferating tumor cells, not in normal tissue.
Despite the apparent preference for tumor cells, propagation of the retroviral vector from producer cells to tumor cells was relatively inefficient, according to Short et al., and only a fraction of the cells in the tumor were infected. Furthermore, practically no .beta.-galactosidase expression was observed when cell-free retroviral vector particles were introduced to a tumor directly rather than in a packaging cell line. Short et al. opined that a packaging cell line might be used to deliver a "killer" or "suppressor" gene to tumor cells, but observed an efficiency of infection far below what would be required for therapeutic utility based on direct gene transduction into all the cells of a tumor.